Recent advances in plant genetic engineering have opened new doors to engineer plants to have improved characteristics or traits. These transgenic plants characteristically have recombinant DNA constructs in their genome that have a protein-coding region operably linked to at least one regulatory region that is the promoter. The promoter can be a strong or weak promoter, or a constitutive or tissue-specific promoter. Besides the promoter, the expression level of the gene product can be modulated by other regulatory elements such as introns. Introns are intervening, non-coding sequences that are present in most eukaryotic genes. Introns have been reported to affect the levels of gene expression. This effect is known as Intron Mediated Enhancement (IME) of gene expression (Lu et al., Mol Genet Genomics (2008) 279:563-572). Callis et al. (Genes Dev. 1987 1:1183-1200) showed that the presence of the first intron from maize alcohol dehydrogenase-1 (Adh1) gene increased the expression levels of transgenes in cultured maize cells up to 100-fold when compared to intronless constructs. Mascarenkas et al. (Plant Mol. Biol., 1990, 15: 913-920) showed that other introns from the maize Adh1 gene could also increase heterologous gene expression in maize protoplasts. Vasil et al. (Plant Physiol., 1989, 91:1575-15790) reported that the constructs containing Shrunken-1 (Sh-1) first intron had much higher expression levels of the reporter gene in plant protoplasts, when compared to the constructs with promoter alone, or to constructs with promoter and Adh-1 first intron. Identifying novel regulatory sequences can lead to finer modulation of gene expression in transgenic plants.
Plant genetic engineering has advanced to introducing multiple traits into commercially important plants, also known as gene stacking. This is accomplished by multigene transformation, where multiple genes are transferred to create a transgenic plant that might express a complex phenotype, or multiple phenotypes. But it is important to modulate or control the expression of each transgene optimally. The regulatory elements such as the promoter and the terminator sequences need to be diverse, to avoid introducing into the same transgenic plant repetitive sequences, which has been correlated with undesirable negative effects on transgene expression and stability (Peremarti et al (2010) Plant Mol Biol 73:363-378; Mette et al (1999) EMBO J 18:241-248; Mette et al (2000) EMBO J 19:5194-5201; Mourrain et al (2007) Planta 225:365-379, U.S. Pat. Nos. 7,632,982, 7,491,813, 7,674,950, PCT Application No. PCT/US2009/046968). Therefore it is important to discover and characterize novel regulatory elements that can be used to express heterologous nucleic acids in important crop species. Diverse regulatory regions can be used to control the expression of each transgene optimally.